不同底物体系下漆酶的失活动力学研究

本文对三种体系的漆酶热失活动力学进行了研究,并对动力学参数进行了相应的分析。结果显示,漆酶在纯水溶液中的失活符合一级动力学模型,其失活方程为lnA=-0. 1353t-2. 2522。在有毒有机物体系(2,4-二氯酚和吲哚溶液)中漆酶的失活仍符合一级失活动力学模型,失活速率常数减小,但半衰期增大。在极性溶液(乙醇)中,漆酶的反应速率常数最小,可能是因为漆酶分子内部作用力的变化导致了漆酶反应变缓;在非极性体系(异辛烷溶液)中,水-疏水体系减少了对酶的伤害,利于酶的稳定,延长了漆酶半衰期;极性或非极性体系中的漆酶失活均符合一级动力学失活模型。     

酵母脂肪酶催化拆分外消旋2-甲基-1-丁醇

近年来,水相中酶催化拆分外消旋酯制备具有光学活性的手性酸或醇,其酶在非水体系的研究取得了重要进展,已发展到用含有微水或几乎无水的有机溶剂取代最初的水相,这样既可避免水参与有关的副反应,又有利于酶的回收。而且酶在某些有机溶剂中比在水中稳定,不会由于微生物污染而使酶失活。脂肪酶能够催化酯的     

脂肪酶不对称水解含酯基的异核金属簇合物

对脂肪酶催化含酯基的异核金属簇合物的水解反应进行了初步研究. 发现脂肪酶对簇合物的酯基进行水解时确有不对称效应, 同时簇骨架中的重金属使酶失活. 尽管不知道水解产物的e.e.值,但从旋光结果可以看出, 酶催化确是一条拆分手性簇合物的途径.     

青霉素酰化酶交联酶聚体的制备及热失活动力学

以0.53 g/mL硫酸铵为沉淀剂, 0.35%(体积分数)戊二醛为交联剂制得青霉素酰化酶交联酶聚体(CLEAs), 酶活收率30.1%, 其最适温度(57 ℃)比游离酶提高10 ℃, 最适pH(10.0)向碱性偏移1.7个单位. 对比游离酶及其CLEAs的热稳定性和热失活动力学模型发现, 游离青霉素酰化酶制成CLEAs后, 其热失活动力学模型由一步失活转变为连串失活, 失活反应活化能由248.8 kJ/mol增加至549.2 kJ/mol, 对CLEAs热稳定性大幅提高的原因进行了解释. CLEAs重复利用7次后, 酶活保留56%以上, 具有良好的重复利用性.     

甘油氢解反应中Pt/WO_3/Al_2O_3催化剂的失活行为（英文）

随着生物柴油产业的高速发展,其副产物甘油大量过剩,亟需寻找高效的甘油高值化利用途径来提高整个生物柴油产业链的经济性.在各种甘油转化路线中,甘油选择性氢解制备1,3-丙二醇可用作高端聚酯PTT的单体,因而成为最具应用潜力的高质化路线之一.同时,该反应涉及到仲羟基的选择性脱除,对其它生物质加氢脱氧反应也具有普遍意义.目前,在提高甘油氢解反应活性和1,3-丙二醇选择性方面已经做了大量的研究工作,但是关于催化剂稳定性的研究却鲜有报道,而后者对于整个过程的工业应用是必不可少的.鉴于此,我们以Pt/WO3/Al2O3为催化剂,在连续运行的固定床反应器上对其甘油氢解反应的长期稳定性以及其失活的机理进行了详细的考察.Pt/WO3/Al2O3催化剂表现出较高的初始活性:甘油的转化率为57.5%,目标产物1,3-丙二醇的选择性可达40.4%,主要副产物1,2-丙二醇、正丙醇和异丙醇的选择性分别为6.3%, 37.3%和11.5%.在长达700 h的稳定性测试中, Pt/WO_3/Al_2O_3催化剂呈现缓慢失活的趋势:甘油转化率从57.5%下降到25.1%,而1,3-丙二醇选择性基本不变.通过对失活曲线进行拟合可以得出催化剂的失活呈现三个阶段:第一阶段内(0-100 h)催化剂的失活速率最快,甘油转化率由57.5%降至44.9%,失活速率为0.111%/h;第二阶段内(100-600 h)催化剂的失活明显变缓,失活速率降至0.037%/h;第三阶段内(600-700 h)甘油转化率基本保持不变.N_2物理吸附、电感耦合等离子体原子发射光谱、热重和程序升温氧化等表征结果显示,活性组分(铂,氧化钨)的流失或积炭都不是催化剂失活的主要原因.同时X射线衍射、高角环形暗场扫描透射电子显微镜和CO化学吸附等表征结果表明,反应过程中Pt/WO_3/Al_2O_3催化剂上Pt颗粒发生了明显的聚集长大,使得Pt的分散度由29.1%降至10.1%.仲丁醇脱水/脱氢探针反应的结果显示,稳定性测试后催化剂在氢气气氛下产生原位B酸的能力显著降低.由此可以得出,反应过程中Pt/WO_3/Al_2O_3催化剂失活的主要原因是催化剂表面Pt颗粒的聚集长大,因而引起催化剂加氢能力的降低,同时Pt-WO_x界面位点的数量下降,使得在氢气气氛下催化剂产生原位B酸的能力大大降低,从而导致了催化剂的失活.     

磷酸锆催化甘油气相脱水制备丙烯醛

以沉淀法、水热合成法和浸渍法制备了磷酸锆催化剂,通过X射线衍射、热重分析、氮气物理吸附、红外光谱和Hammett指示剂法对催化剂进行了表征,并将该催化剂用于甘油气相脱水反应.研究表明,由沉淀法得到的磷酸锆经过400℃焙烧后能达到最佳催化活性,在温和条件下,甘油可完全转化,丙烯醛选择性为81%,反应24h内催化剂失活不明显.不同方法制备的磷酸锆其结构和表面酸性显著不同,催化剂表面酸性对催化剂活性、丙烯醛选择性和催化剂的寿命均有较大影响.     

基于聚多巴胺的磁性纳米生物催化剂高效催化二氢杨梅素酰化

脂肪酶是一种三酰基甘油水解酶,目前广泛用于油脂化学、食品、有机合成和生物医药等领域.但是,游离脂肪酶在有机反应体系中容易失活,难以从反应体系中回收,导致其循环利用困难和生产成本增加.因此,需要对游离脂肪酶进行固定化,提高酶的稳定性和重复使用性,使其能够大规模用于工业生产.
　　磁性四氧化三铁纳米粒子(MNPs)具有其超顺磁性和大比表面积等性质,但MNPs需表面修饰才能进一步应用.近年来,仿生矿化法制备的聚多巴胺纳米材料受到人们关注.在仿生矿化过程中,单体多巴胺经自聚合作用后形成聚多巴胺,该反应活性高,能对各类有机和无机纳米材料进行表面修饰.而且,聚多巴胺表层中的活性基团能与含有氨基和巯基的生物大分子发生迈克尔加成或席夫碱反应,从而将生物大分子固定在材料表面.
　　本文利用聚多巴胺表面修饰MNPs,对所得聚多巴胺表面修饰的四氧化三铁纳米粒子(PD-MNPs)进行了结构表征.结果表明, PD-MNPs尺寸在14 nm左右.同时,成功将黑曲霉脂肪酶(ANL)固定在PD-MNPs上,结果显示在pH=8、固定化时间为12 h条件下,酶负载量为138 mg/g,酶活回收率达到83.6%,而且固定化酶的pH稳定性及热稳定性、储藏稳定性都优于游离酶.动力学研究表明,固定化酶Km值(63.2 mmol/L)低于游离酶(74.5 mmol/L),固定化酶的底物亲和性增强.进一步研究了固定化酶和游离酶在乙腈、二甲基亚砜、乙醇和[HMIm]BF4这四种溶剂中的溶剂耐受性,结果显示固定化酶的耐受性均强于游离酶.采用红外光谱对游离酶和固定化酶二级结构的分析表明,游离黑曲霉脂肪酶经固定化后,α-螺旋和β-折叠含量分别增加了0.84%和2.74%,使得固定化后α-螺旋和β-折叠中存在的氢键能够更好地保持酶结构刚性,避免因结构改变而引起酶失活,增强了固定化酶在溶剂中的耐受性.
　　二氢杨梅素是一种具有类黄酮结构的天然产物,具有抗氧化、抗菌、抗肿瘤和保护肝脏等作用,但其脂溶性很差,很难透过细胞膜被人体吸收.本课题组曾首次以乙酸乙烯酯为酰基供体,采用游离脂肪酶生物催化方法成功将二氢杨梅素酰化.本文考察了PD-MNPs固定化脂肪酶在二氢杨梅素酰化反应中的应用.结果表明,与游离酶相比,固定化酶在反应介质二甲基亚砜中的耐受性更强,反应48 h后其催化二氢杨梅素酰化的转化率接近80%,明显好于游离酶(69%).固定化酶催化二氢杨梅素酰化的最适底物摩尔比、温度和酶量分别为10：1(乙酸乙烯酯：二氢杨梅素)、45oC,和40 U.此外,固定化酶在外界磁场作用下能迅速从反应混合物中分离,从而可回收利用,在重复使用10次后,其活性仍保持在初始活性的55%以上,具有良好的工业应用前景.     

生物相容性嵌段型聚电解质胶束在水溶液中的酶降解行为

采用激光光散射仪和原子力显微镜研究了生物相容性嵌段型聚电解质聚左旋乳酸-b-聚甲基丙烯酸二甲氨基乙酯(PLLA-b-PDMAEMA)胶束在水溶液中2个温度(室温25.0℃和人体温度36.8℃)和2个pH值(肿瘤pH=4.9和正常组织pH=7.4)条件下的酶降解行为. 酶降解过程中存在一个失活时间, 在此之前, 胶束的酶降解遵循逐个降解机理. 失活时间之后, 出现裂纹或是通道的胶束核为降低其在溶剂中的表面积, 从而降低体系自由能, 胶束之间发生了聚集. 升高温度后, 酶的活性提高, 初始降解速率加快. 由于pH=4.9时胶束壳层因静电斥力作用而较为伸展, 使得胶束降解更快.     

蒸汽失活裂化催化剂沸石活性组元的再活化研究

研究了蒸汽失活的裂化催化剂的再活化过程.高温水蒸汽下失活的含Y型沸石的裂化催化剂在碱性水热条件下进行原位晶化处理并经过含铵离子的水溶液交换以恢复其裂化活性,在整个处理过程中保持催化剂的粒度不变.对含ZSM-5类沸石的失活裂化催化剂则采用含VA族元素的铵盐溶液处理,并在350℃～600℃的温度范围内进行焙烧来恢复其择形裂解活性.     

多孔材料固定化脂肪酶的研究进展

在有机合成中,脂肪酶是一种符合绿色化学理念、能显著提高催化效率和对生化工业具有重要意义的生物催化剂,它的研究和应用涉及很多领域.然而,在众多有机反应中,脂肪酶容易受水、温度、 pH值、酶液浓度、底物浓度、酶的激活剂或抑制剂等许多因素的影响,导致失活,产率降低.为了解决这一问题,酶的固定化技术引起了广大科研工作者的浓厚兴趣,并发现了很多酶的固定化载体.其中,多孔材料类固定化载体颇受青睐,它具有孔隙率高、比表面积大、相对密度低、吸附性能较佳、渗透性能较好和精确的分子识别功能等优点.实验证明,多孔材料固定化酶比游离酶的应用效果更佳,多次循环利用后仍旧保持较高的酶活性.我们主要对多孔材料在固定化脂肪酶方面的应用和固定化酶的催化效果做了一个总结,多孔材料主要包括纳米多孔材料、大配体多孔材料、碳骨架多孔材料、氧化硅骨架多孔材料、聚合物类多孔材料等.     

Influence of microwave radiation on free Candida antarctica lipase B activity and stability

The influence of microwave heating on free Candida antarctica lipase B activity and stability was studied over the temperature range from 40 to 110 degrees C. Concerning the lipase activity, identical initial rate and conversion yield were obtained under microwave radiation and classical thermal heating for the alcoholysis between ethyl butyrate and butanol in a solvent-free system. On the other hand, the kinetics of the free lipase inactivation in butanol appears to be influenced by the heating mode. The Arrhenius plot obtained under classical heating was linear over all the temperature range studied whereas a biphasic Arrhenius plot was obtained under microwaves. The non-classical effect of the microwave heating on the initial rate of the enzymatic inactivation was thus dependent on the temperature of incubation.     

Thermostability of Cromobacterium viscosum lipase in AOT/isooctane reverse micelle

The thermostability of Cromobacterium viscosum lipase (EC 3.1.1.3) entrapped in AOT (sodium bis-[2-ethylhexyl] sulfosuccinate) reverse micelles was increased by the addition of short-chain polyethylene glycol (PEG 400). Two different approaches were considered: (1) the determination of half-life time and (2) the mechanistic analysis of deactivation kinetics. The half-life of lipase entrapped in AOT/isooctane reverse micelles with PEG 400 at 60 degrees C was 28 h, ninefold higher than that in reverse micelles without PEG 400. The lipase entrapped in both reverse micellar systems followed a series-type deactivation mechanism involving two first-order steps. The deactivation constant for the first step at 60 degrees C in PEG containing reverse micelles was 0.055 h!1, 11-fold lower than that in reverse micelles without PEG, whereas it remained almost constant for the second step. The inactivation energy of the lipase entrapped in reverse micelles with and without PEG 400 was 88.12 and 21.97 kJ/mol, respectively.     

Enzymatic conversion of waste cooking oils into alternative fuel—Biodiesel

Production of biodiesel from pure oils through chemical conversion may not be applicable to waste oils/fats. Therefore, enzymatic conversion using immobilized lipase based on Rhizopus orzyae is considered in this article. This article studies this technological process, focusing on optimization of several process parameters, including the molar ratio of methanol to waste oils, biocatalyst load, and adding method, reaction temperature, and water content. The results indicate that methanol/oils ratio of 4, immobilized lipase/oils of 30 wt% and 40°C are suitable for waste oils under 1 atm. The irreversible inactivation of the lipase is presumed, and a stepwise addition of methanol to reduce inactivation of immobilized lipases is proposed. Under the optimum conditions the yield of methyl esters is around 88–90%.     

Thermal decomposition of polyurethane elastomers containing antipyrene

Thermal analysis, IR and emission spectroscopy were used to study the influence of phosphorus and phosphorus/chlorine-containing antipyrenes on the thermal decomposition of polyurethane elastomers based on 4,4′-diphenylmethane diisocyanate and polytetraoxymethylene. The introduction of P-containing antipyrene into the structure of polyurethane elastomers induces changes in the mechanism and kinetics of thermal decomposition towards inactivation of the exothermal reactions of oxidative decomposition. It causes a shift of the first exothermal peak towards higher temperatures by 100° and helps the formation of temporarily stabilized structures. P/Cl-containing antipyrene has a smaller positive effect towards inactivation of the exothermal decomposition reactions and the formation of temporarily stabilized structures.     

Investigation of water influence on coal based on thermal oxidative degradation kinetics

Journal of Thermal Analysis and Calorimetry - In order to further understand the effect of the water content on spontaneous combustion of coal, the thermal oxidative degradation kinetics of coals...     

Biocatalytic Gelation of Silica in the Presence of a Lipase

The effect of the lipase from Burkholderia cepacia (previously known as Pseudomonas cepacia) on the gelation kinetics and gel structure was examined on a type of silica aerogel made from a mixture of methyltrimethoxysilane and tetramethoxysilane. For this purpose, gels were made with increasing concentrations of lipase in otherwise constant other conditions (pH, water and Si precursors concentrations). It was found that the enzyme accelerated the gelation kinetics, hence was participating in some way to the hydrolysis of the silica precursor. The structure of the gel was simultaneously modified to produce an increasing proportion of Q⁴ silicon sites.     

微乳液中脂肪酶和含明胶的微乳液凝胶中固定化脂肪酶的催化特性

在双2-乙基己基琥珀酸酯磺酸钠(AOT)油包水微乳液中Calytical脂肪酶催化月桂酸和戊醇的酯化反应动力学研究表明,反应符合乒乓(BiBi)机制.表观速率常数k_m酸=0.13518mol/L,k_m醇=0.22423mol/L,最大反应速度v_max=1.3873×10^-5mol/(L·min·mg).将该脂肪酶固定于含明胶的微乳液凝胶(MBGs)中,制得固定化脂肪酶,含酶MBGs在非极性溶剂中可作为固相催化剂,并研究了其在辛烷中催化酯化的性能.所制得的含酶MBGs物理稳定性好,重复利用10次以上,其转化率仍达初始转化率的90%.     

Immobilization of Yarrowia lipolytica Lipase—a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques

Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.     

Lipases at interfaces: unique interfacial properties as globular proteins

The adsorption behavior of two globular proteins, lipase from Rhizomucor miehei and beta-lactoglobulin, at inert oil/water and air/water interfaces was studied by the pendant drop technique. The kinetics and adsorption isotherms were interpreted for both proteins in different environments. It was found that the adopted mathematical models well describe the adsorption behavior of the proteins at the studied interfaces. One of the main findings is that unique interfacial properties were observed for lipase as compared to the reference beta-lactoglobulin. A folded drop with a "skinlike" film was formed for the two proteins after aging followed by compression. This behavior is normally associated with protein unfolding and covalent cross-linking at the interface. Despite this, the lipase activity was not suppressed. By highlighting the unique interfacial properties of lipases, we believe that the presented work contributes to a better understanding of lipase interfacial activation and the mechanisms regulating lipolysis. The results indicate that the understanding of the physical properties of lipases can lead to novel approaches to regulate their activity.     

Effect of polyvinylalcohols on the thermostability of lipase fromCandida rugosa

Lipase fromCandida rugosa was stabilized against thermal inactivation in the presence of polyvinylalcohols (PVA) of different molecular weights. The apparent rate constant of the lipase inactivation, k_d, at 49°C is 0.049/min and 0.022/min in the absence and in the presence of PVA (mol wt 22,000), respectively. The improvement of the lipase thermostability by adding PVA was confirmed by differential scanning calorimetry. The presence of PVA had also an effect on the hydrolytic activity of the enzyme. Furthermore, lipase was modified by covalent linkage to PVA by means of an original procedure. With respect to the native enzyme, the modified lipase has a slightly lower specific activity, but it is more stable against heat denaturation (k _d 0.032/min at 49°C).